An Ayurvedic Herbal Extract Inhibits Streptococcus mutans Biofilm Formation and Disrupts Preformed Biofilms in vitro

Objective: Sudantha® (SUD), a natural proprietary mixture of herbal extracts that has been incorporated into toothpaste, has been shown in two separate placebo controlled human clinical studies to promote gingival health; and reduce gingival bleeding and plaque formation. However, the herbal based anti-gingivitis mechanisms of Sudantha are not fully understood. The objective of this study was to determine the effect of Sudantha on dental plaque biofilms by investigating its effect on mono-culture biofilms of a primary colonizer, Streptococcus mutans, in vitro. Results: This study found that SUD contributes to the maintenance of oral health through the inhibition of S. mutans biofilm formation. In addition, SUD disrupted preformed S. mutans biofilms after exposure to SUD for 4 hours. Together, this pilot data suggests the inhibition of S. mutans biofilm formation and disruption represents one potential mechanism by which the herbal extract is able to reduce the oral bacterial biofilm resulting in its effective against gingivitis and its potential use in countering biofilm associated oral disease.


Introduction
Streptococcus mutans is a normal constituent of the human oral microbiota and is considered to be a primary etiological agent in caries and a primary colonizer of dental biofilms [1,2]. It is an aciduric, facultative anaerobic gram positive cocci equipped with an array of virulence traits such as the ability to adhere to tooth surfaces and form biofilms. Acidogenecity and acidurity, caused by the lactic acid byproducts of sucrose metabolism by S. mutans, are This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. significant factors in the etiology and pathogenesis of dental caries [1]. As a commensal member of the oral cavity in health, complete elimination of this organism would not be strategic from an ecological balance point of view. There is increasing recognition that the preservation and/or restoration of the health-associated microbiome are critical to maintain health rather than complete elimination of specific members of the ecosystem in diseased sites or individuals [3,4]. Therefore caries prevention strategies has shifted focus towards targeting virulence factors rather than in the elimination of pathogenic bacteria due to inherent problems associated with antimicrobials such as non-discriminating action affecting all bacteria, development of resistance and superinfections. In a broader sense, it is possible that the use of anti-virulence strategies may exert less stress on the bacteria in the ecosystem so that the drive to develop resistant strains would be reduced. Hence, investigators have directed their efforts towards identification of anti-virulence factors to reduce carcinogenicity of this bacterium.
A variety of natural compounds isolated from plants have been shown to possess activity against major bacterial virulence factors such as quorum sensing, biofilms, motility, toxins and enzymes [5,6]. Sudantha® (SUD) is toothpaste containing a proprietary Ayurvedic blend of plant extracts, which has been used traditionally for oral care [7][8][9][10][11][12][13]. In a recently published study it was found that SUD differentially modulated the gingival epithelial cell interleukin-8 (IL-8) response to bacteria and host cytokines [14]. It was postulated that this host associated inflammatory modulation activity of the SUD extract may be contributing to its clinical efficacy in maintaining gingival health as observed in two previously published clinical trials [15,16]. The objective of this study was to determine if SUD also had an effect on dental plaque accumulation separate from the host effects previously published [14]. Streptococcus mutans is the principal bacterium associated with dental caries and its accumulation in the supra-gingival plaque biofilm is strongly associated with the development of dental caries (1). Moreover, streptococci are considered pioneer species that allow for the subsequent sequential assembly of oral bacteria to form plaque biofilms, which can initiate diseases such as caries, gingivitis, and periodontitis [1,17]. Therefore, the ability of the SUD extract to inhibit S. mutans biofilm formation was determined.
This study found that SUD was able to inhibit S. mutans biofilm formation and disrupt S. mutans pre-formed biofilms. These data demonstrate a potential beneficial activity of SUD in the prevention of S. mutans biofilm mediated bacterial accumulation and warrants further investigation.

Sudantha herbal extract
Sudantha (SUD) extract is the active ingredient of a commercially available tooth paste Sudantha. It is a standardized crude proprietary mixture of 9 herbs formulated by a specialist panel of Ayurvedic clinicians and is quality controlled by high performance liquid chromoatography (HPLC). Its ingredients are: heartwood of cutch tree (Acacia chundraWilld.), malabar nut leaf (AdhatodavasicaNees.), Spanish cherry bark (Mimusopselengi L.), black pepper (Piper nigrum L.), pongam oil tree root (Pongamiapinnata(L.) Pirerre), Aleppo oak galls (Quercusinfectoria Olivier.), clove (Syzygiumaromaticum L.), myrobalan fruit (Terminaliachebula Retz.), and ginger (Zingiberofficinale Roscoe). This medicinal extract was stored at 4°C in the dark and freshly prepared to a stock concentration of 120 mg/ml a 12% ethanol diluted with 1% sucrose TYK medium (TYK+S). This stock concentration was then subsequently serially diluted with TYK +S medium to produce working experimental concentrations.

Biofilm inhibition
Starter cultures were grown with indicated concentrations of SUD in TYHK+S or TYK+S medium in 96-well flat bottomed plates in triplicate overnight (16-18 hours) at 37°C under anaerobic conditions; after which, crystal violet and susceptibility assays were performed. Wells with media alone served as positive controls and were used as the reference for total biofilm inhibition calculations, while wells without bacteria served as negative controls.

Susceptibility assays
Total contents of the wells (planktonic and biofilm cells) were removed and resuspended in PBS. Then, ten-fold serial dilutions were plated onto blood agar plates and incubated at 37°C under anaerobic conditions for 24 hours before counting bacterial colonies. This experiment was performed 5 independent times in triplicate.

Biofilm disruption assays
For biofilm disruption studies, starter cultures of S. mutans in TYK +S medium were grown in 96-well flat bottomed plates overnight at 37°C under anaerobic conditions. Afterwards the medium was discarded and replenished with indicated concentrations of SUD diluted in TYK+S medium in triplicates. Plates were then incubated for an additional 4 hours at 37°C under anaerobic conditions and crystal violet assays were performed. Biofilms grown in TYK+S alone served as positive controls and were used as the reference for total biofilm inhibition calculations, while wells without bacteria served as negative controls.

Crystal violet assay
Biofilms were washed three times with deionized water to remove non-adherent cells. Then 0.1% crystal violet dye was added and incubated at room temperature for 10 minutes. Following incubation, the dye was removed and biofilms were washed three times with deionized water. Biofilms were then allowed to air dry completely and 30% acetic acid solution was added for 15 minutes. Solubilized dye was then mixed thoroughly and transferred into a new plate and read at an OD of 575 nm using a microplate reader. Percent biofilm inhibition was calculated by Percent biofilm inhibition = Control OD 575 − Test OD 575 /Control OD 575 × 100 %

Statistical analysis
Unpaired Student T-test was performed to determine significance for percent biofilm inhibition.

SUD inhibits initial Streptococcus mutans biofilm formation
To assess the ability of SUD to inhibit S. mutans biofilm formation, planktonic S. mutans was grown overnight with half serial dilutions of SUD with the highest concentration at 3.75 mg/ml and lowest at 0.06 mg/ml. Overnight exposure to SUD showed dose dependent biofilm inhibition, where significant inhibition at 42% compared to control biofilms grown in media alone, occurred at 0.23 mg/ml SUD (p=0.002) and peak inhibition occurred around 92% at 0.94 mg/ml SUD (p<0.0001) ( Figure 1A). Concentrations above 0.94 mg/ml SUD did not proceed to inhibit biofilm formation any further.
Enumeration of S. mutans cells, both biofilm and planktonic, exposed to 0.6-2.5 mg/ml SUD and grown overnight in a similar manner revealed a trend of dose dependent decrease in the total number of colony forming units (CFU) ( Figure 1B). This suggests that inhibition of biofilms observed in ( Figure 1A) may be partially due to reduction of cell viability.

SUD disrupts pre-formed Streptococcus mutans biofilms
Subsequent to determining that SUD inhibited S. mutans biofilm formation, we assessed whether SUD also had the capability to disrupt pre-formed S. mutans biofilms. S. mutans biofilms grown overnight in 96-well plates were exposed to various concentrations of SUD for 4 hours. SUD disrupted pre-formed biofilms in a dose dependent manner, although biofilm release required higher concentrations of SUD compared to concentrations required for biofilm inhibition (Figure 2). Biofilm disruption was greatest with 3.75 mg/ml SUD which produced a 30% reduction in biofilm after 4 hours compared to controls with media alone (p<0.0001). The lower concentrations of 1.88 mg/ml and 0.94 mg/ml SUD examined did not show significant reduction in biofilms.

Discussion
Streptococcus mutans is equipped with an array of virulence factors which help facilitate its adhesion, biofilm formation, and survival in a highly acidic environment [2,18]. As primary colonizers, streptococci play a large role in the formation of dental plaque, which is involved in the etiology of caries and periodontal diseases [1,17]. Of the several secreted products of S. mutans, glucosyltransferases (gtfs) and their glucan products are major virulence factors and play a significant role in biofilm production [18]. Glucosyltransferases B, C and D of S. mutans produce insoluble and soluble exopolysaccharides (glucans), a major component of the biofilm matrix, utilizing dietary sucrose as their primary substrate [18]. The collective action of these products enable bacteria in biofilms to survive harsh conditions such as mechanical shear forces, temperature extremes, physical properties of saliva, and exposure to antimicrobial agents in toothpastes and mouthwashes, as well as to antibiotics taken systemically.
Both in vitro and in vivo studies have reported successful application of herbal extracts and plant derived polyphenols on reduction of oral bacterial counts and inhibition of biofilm production [14,15,[18][19][20][21][22][23]. Plant derived polyphenols are a class of natural compounds, including flavonoids, phenolic acids, and tannins, produced by plants in defense against invading pathogens and have been shown to inhibit S. mutans synthesis of glucan polysaccharides through the suppression of gtf genes [21]. Polyphenols have also been reported to be effective against biofilms produced by other oral pathogens, such as Porphyromonas gingivalis and Enterococcus faecalis, which are involved inflammatory diseases of the gums and root canal infections, respectively [22,23].
This pilot study found that SUD was able to inhibit initial S. mutans biofilm formation, as well as disrupt pre-formed biofilms. Biofilm inhibition was observed to be statistically significant with overnight exposure to 0.23 mg/ml to 3.75 mg/ml SUD. It is roughly estimated that an individual using a pea-sized amount of Sudantha® toothpaste will be exposed to 37.5 mg/ml SUD, which will be further diluted 10-fold with saliva in the mouth to ~3.75 mg/ml.Therefore, biofilm inhibition was observed at a fraction of the working peasized toothpaste concentration. Furthermore, reduction in total bacterial counts (planktonic and biofilm cells) after exposure to SUD overnight compared to controls suggest that initial biofilm inhibition may be partially due to antibacterial effects of the extract, which may occur as a direct or indirect effect of SUD. Alternatively, it is feasible that SUD may indirectly effect bacterial survival by suppression of gtf genes involved in exopolysaccharide production placing S. mutans into a more vulnerable state. This possibility will be examined in subsequent study.
Compared to biofilm inhibition, preformed biofilm disruption required higher concentrations of SUD at 3.75 mg/ml when exposed for 4 hours. The higher concentration required for disruption may be due to the decreased exposure time.

Conclusion
Altogether, our data demonstrates that SUD is an effective agent against S. mutans biofilm in vitro. This data may partially explain one potential mechanism behind the efficacy of SUD gingival health that was observed in two randomized clinical trials by the significant reduction of gingival bleeding, dental plaque biofilm formation, and anaerobic bacterial counts. Furthermore, in vitro studies showed the ability of SUD to modulate host IL-8 inflammatory responses. Collectively, the data from this pilot study adds to the line of evidence behind the effectiveness of SUD in the maintenance of oral health and deserves further investigation.

Limitations
The effect on mixed species biofilms was not examined and therefore these experiments address intra and not inter species adhesive interactions. Furthermore, due to the blended characteristic of the Sudantha® extract, it is difficult to assess which individual component within this mixture is mechanistically responsible for the anti-biofilm properties.